The present invention is directed to methods to alter secondary metabolism of a plant, more specifically phenylpropanoid metabolism. The present invention is also directed to novel mutant polynucleotide molecules, referred to as ref8, that encode an Arabidopsis p-coumarate 3-hydroxylase having altered biological activity. The present invention is also directed to uses of the novel nucleotide sequences set forth herein, including their use in vectors and other DNA constructs for transforming plants and microorganisms. The DNA constructs and transgenic plants are further aspects of the present invention.
The publications, patents and other materials used herein to illuminate the background of the invention, and in particular cases, to provide additional details respecting the practice, are incorporated by reference, and for convenience are referenced in the following text by author and date and are listed alphabetically by author in the appended bibliography.
By way of background, C3H is an enzyme of the phenylpropanoid pathway. Phenylpropanoid compounds have a wide array of important functions in plants. They serve in the interaction of plants with their biotic and abiotic environments, mediate certain aspects of plant growth and development, and are important structural components of the plant secondary cell wall. For example, stilbenes and isoflavones are important phytoalexins in plants (Nicholson and Hammerschmidt, 1992). In maize and petunia, flavonoids have been shown to be necessary for pollen viability (Coe et al., 1981; Taylor and Jorgensen, 1992; van der Meer et al., 1992), and have been suggested to be endogenous modulators of auxin transport (Mathesius et al., 1998). Hydroxycinnamic acids lead to the synthesis of UV-sunscreens in plants (Landry et al., 1995), and are also precursors for lignin (Lewis and Yamomoto, 1990). Simpler phenylpropanoid-derived molecules such as acetosyringone act as signaling molecules in the interaction of plants with Agrobacterium (Stachel et al., 1985). Lignan glycosides known as dihydrodiconiferyl glycosides (DCGs) have cytokinin-like activity in plants (Binns et al., 1987; Lynn et al., 1987; Teutonico et al., 1991; Orr and Lynn, 1992), and may be responsible for growth abnormalities seen in some transgenic plants in which phenylpropanoid metabolism has been perturbed (Tamagnone et al., 1998). Phenylpropanoids are also increasingly being recognized as having an impact on human health. For example, isoflavones and lignans have beneficial estrogen-like activity in humans which is prompting their use as neutraceuticals (Bingham et al., 1998) and the stilbene resveratrol is thought to provide the health benefits associated with moderate wine consumption (Jang et al., 1997). All of the above examples make a compelling argument for improving our understanding of phenylpropanoid metabolism and its regulation.
Advances in biotechnology have provided the tools with which to manipulate phenylpropanoid metabolism, and a number of cases have illustrated the potential value of this approach. The capacity to synthesize resveratrol has been transferred to tobacco by transformation with a construct encoding stilbene synthase (Hain et al., 1993). Flower pigmentation has been successfully manipulated in petunia by introduction of the maize gene encoding dihydroflavonol reductase (Meyer et al., 1987). Similarly, novel and valuable varieties of cut flowers are being generated by introduction of the gene encoding flavonoid 3′,5′-hydroxylase which leads to the accumulation of blue trihydroxy-substituted anthocyanins (Holton et al., 1993). The manipulation of lignin biosynthesis has also been extensively investigated, with results ranging from substantial decreases in total lignin content to dramatic changes in lignin monomer composition (Meyer et al., 1998). As additional targets for the metabolic engineering of phenylpropanoid metabolism are investigated, their manipulations may lead to plants with enhanced nutritional value, crops that synthesize large amounts of secondary metabolites for industrial use, the modification of lignin quality and quantity in plants, and plants with enhanced UV tolerance. For these approaches to be successful, it is essential that we have a thorough knowledge of all of the catalysts involved.
Most of the genes encoding the enzymes of the phenylpropanoid pathway have been cloned over the last ten years by standard biochemical approaches, and since their original isolation, an array of orthologues have been cloned from various species. These include the genes encoding caffeoyl CoA O-methyltransferase (CCoAOMT), cinnamate 4-hydroxylase (C4H), cinnamyl alcohol dehydrogenase (CAD), cinnamoyl CoA reductase (CCR), 4-(hydroxy) cinnamoyl CoA ligase (4CL), phenylalanine ammonia-lyase (PAL), and caffeic acid/5-hydroxyferulic acid O-methyltransferase (COMT). The two cytochrome P450-dependent monooxygenases (P450s) in the pathway, C4H and ferulate 5-hydroxylase (F5H) were more difficult targets because the instability, low abundance, and membrane-bound nature of plant P450s makes conventional purification problematic. Despite these difficulties, the gene encoding C4H was recently identified (Mizutani et al., 1993b; Teutsch et al., 1993) following purification of the enzyme (Gabriac et al., 1991; Mizutani et al., 1993a). Because the activity of F5H had been detected only once in plant extracts (Grand, 1984), and because F5H proved unstable to purification, the detailed characterization of F5H was made possible only through the genetic analysis of the Arabidopsis fahl mutant (Chapple et al., 1992). Using this mutant, the gene encoding F5H was cloned by T-DNA tagging, an approach that circumvented the requirement of protein purification (Meyer et al., 1996).
The biosynthesis of many phenylpropanoids requires two distinct hydroxylation steps. C4H introduces the first hydroxyl group at the 4-position of the aromatic ring of cinnamic acid. C4H activity is readily measured in plants, and was one of the first plant enzymes to be recognized to be a P450. The next hydroxylation occurs at the 3-position of the ring, and is necessary for the synthesis of many important phenylpropanoid compounds. In contrast to C4H, the 3-hydroxylase of the phenylpropanoid pathway has not been fully characterized. The enzyme that catalyzes this reaction is known as p-coumarate 3-hydroxylase (C3H), although this hydroxylation may also be carried out at the CoA thioester level by p-coumaroyl CoA 3-hydroxylase (pCCoA3H). It is not clear which of these two activities is relevant to phenylpropanoid metabolism because the 3-hydroxylase is an enigmatic enzyme. It has eluded attempts over the last thirty years to unambiguously characterize it in detail at the enzymatic level. It was the last gene of the phenylpropanoid pathway to be cloned.
Over the past thirty years, many researchers have attempted to assay, characterize and purify C3H. C3H activity has been detected in extracts of spinach beet, sorghum, oak, mung bean, and potato (Vaughan and Butt, 1969; Vaughan and Butt, 1970; Alibert et al., 1972; Bartlett et al., 1972; Stafford and Dresler, 1972; Halliwell, 1975; Duke and Vaughn, 1982; Bolwell and Butt, 1983; Boniwell and Butt, 1986; Kojima and Takeuchi, 1989). C3H has been characterized as a copper-containing mixed function oxidase (Vaughan and Butt, 1970) that requires an electron donor for activity. In most cases ascorbate has been found to be the optimal donor, although it is often required in very high concentration with Km values as high as 10 mM (Kojima and Takeuchi, 1989). NADPH and 2-amino-4-hydroxy-6,7-dimethylpteridine also served as a reductant in some cases (Vaughan and Butt, 1970; Stafford and Dresler, 1972), whereas other enzyme preparations showed an absolute requirement for FAD or FMN (Boniwell and Butt, 1986). C3H has been reported to be associated with the chloroplast thylakoid membranes, where it was suggested that plastoquinone or ferredoxin could serve as the electron donor in vivo (Bartlett et al., 1972).
In most experiments, C3H activity was associated with a phenolase activity which oxidizes dihydroxyphenols to their corresponding orthoquinones. In some cases, C3H activity could be purified away from phenolases, but generally the semi-purified C3H preparations retained substantial ability to oxidize dihydroxyphenols (Stafford and Dressler, 1972). Still other experiments were aimed at correlating light- and wound-induced increases in PAL and C4H with induction of putative C3H activities (Bolwell and Butt, 1983). Once high background levels of phenolase were accounted for, some increases in C3H activity could be identified, and although the corresponding protein was partially purified it was not studied further. In experiments using mung bean seedlings treated with the fungal toxin tentoxin, phenolase activity was completely eliminated while the accumulation of caffeic acid in vivo and in vitro remained unaffected. These experiments provided strong evidence that distinguished C3H from phenolase (Duke and Vaughn, 1982).
Other research has suggested that the 3-hydroxylation reaction occurs at the level of p-coumaroyl esters such as p-coumaroyl quinate, p-coumaroyl shikimate, or p-coumaroyl glucose (Heller and Kühnl, 1985; Kühnl et al., 1987; Tanaka and Kojima, 1991). Based upon their association with membranes and classical inhibitor studies, the first two activities were attributed to P450s. The latter enzyme appeared to be closely related to the aforementioned phenolases and its involvement in phenylpropanoid biosynthesis has been viewed skeptically by some authors (Wang et al., 1997).
Finally, another body of work suggests that 3-hydroxylation occurs at the level of the CoA thioester, and that the product of this reaction is used both as a primer for dihydroxylated anthocyanin biosynthesis, and as an acyl-donor. In Silene dioica, the P gene controls hydroxylation of the 3′ position of the anthocyanin B ring and the substitution pattern of the acyl-moiety esterified to the anthocyanin (Kamsteeg et al., 1980). Wild-type anthocyanins are caffeic acid esters of dihydroxy-substituted cyanidin glucosides, while homozygous p mutants accumulate monohydroxylated pelarogonidin glucosides that are esterified with p-coumaric acid. In this system, the pCCoA3H activity was shown to be an NADPH-dependent monooxygenase, and this activity was shown to be absent in p/p petal extracts (Kamsteeg et al., 1981). The generality of these findings in relation to flavonoid synthesis is in doubt, however, since in other systems flavonoid hydroxylation occurs at the dihydroflavonol level and is catalyzed by specific P450s (Holton et al., 1993; Brugliera et al., 1999). A Zn2+— and ascorbate-dependent pCCoA3H has also been assayed in elicitor-induced cultures of parsley cells (Kneusel et al., 1989). The activity of this enzyme was shown to be highly sensitive to pH, and this was suggested to be a mechanism for enzyme activation in response to elicitation. While the nature and identity of pCCoA3H remains questionable, the presence of CCoAOMT in plants (Pakusch et al., 1989; Schmitt et al., 1991; Ye et al., 1994; Ye and Varner, 1995), and the recent demonstration that its activity contributes substantially to lignin biosynthesis (Zhong et al., 1998) suggests that pCCoA3H activity may be relevant to phenylpropanoid metabolism.
The potential success or failure of metabolic engineering efforts hinge upon a thorough understanding of the target pathway. Similarly, the ability to interpret data from experiments that examine plant responses to pathogen or herbivore attack depends upon a comprehensive understanding of the metabolic framework that underlies those responses. One example that is particularly relevant to this proposal can be found in the recent rewriting of the phenylpropanoid pathway that has been the unexpected outcome of experiments aimed at the modification of lignin content and composition.
The longstanding model of phenylpropanoid metabolism has postulated a branched but linear pathway (Higuchi, 1981). According to this model, the phenylpropane skeleton of phenylalanine is converted to hydroxycinnamic acids which serve as precursors for flavonoids, lignin and hydroxycinnamic acid esters. More recently, a different route for the biosynthesis of lignin monomers has received attention (Kneusel et al., 1989; Kühnl et al., 1989; Pakusch et al., 1989; Pakusch et al., 1991; Schmitt et al., 1991; Ye et al., 1994; Ye and Varner, 1995; Zhong et al., 1998). This so-called “alternative pathway” involves the activation of p-coumaric acid to its coenzyme A thioester, followed by hydroxylation and methylation reactions that ultimately generate feruloyl-CoA. Considering that ferulic acid can also be synthesized by the free acid pathway and can be activated to its CoA thioester by 4CL, lignin monomer biosynthesis probably occurs via a cross-linked network of pathways. Indeed, the continued accumulation of guaiacyl lignin in COMT suppressed plants (Atanassova et al., 1995; Van Doorsselaere et al., 1995) indicates that the alternative pathway is a major contributor to lignin biosynthesis in woody plants. This hypothesis has been tested directly by the generation of transgenic tobacco downregulated for caffeoyl-CoA O-methytransferase (CCoAOMT) activity (Zhong et al., 1998). These plants had lower total lignin content, demonstrating that the alternative pathway is a quantitatively important route for monolignol biosynthesis and that COMT activity cannot compensate for a decrease in the expression of CCoAOMT.
In addition to the incorporation of the “alternative pathway”, data from the present research and that of others has necessitated a further revision of the lignin biosynthetic pathway (Humphreys, et al., 1999; Osakabe, et al., 1999). In these experiments, F5H expressed in yeast demonstrated Michaelis-Menten kinetics with regard to ferulate hydroxylation with a Km of 1 mM and a Vmax of 4 pKat mg−1 protein. This Km was unexpectedly high considering that C4H, a P450 three steps earlier in the pathway, exhibits a 4 μM Km for its substrate (Urban et al., 1994). This inconsistency led us to test the hypothesis that phenylpropanoid pathway intermediates other than ferulate might be better substrates for F5H. Assays conducted with coniferaldehyde demonstrated that the Km and Vmax of F5H for this substrate were 1 μM and 5 pKat mg−1 respectively, and the corresponding values for coniferyl alcohol were 3 μM and 6 pKat mg−1. These data strongly suggest that coniferaldehyde and coniferyl alcohol are the preferred substrates for F5H, and that F5H probably acts later in the pathway than was previously envisioned. Other experiments have also suggested that COMT is actually a 5-hydroxyconiferyl alcohol/5-hydroxyconiferaldehyde O-methyltransferase that acts immediately downstream of F5H in the lignin biosynthetic pathway (Humphreys et al., 1999; Osakabe et al., 1999; Li et al., 2000).
The experiments described above, among others, have demonstrated that understanding of phenylpropanoid metabolism is still incomplete. Although plant secondary metabolism has been studied for many decades, modern molecular, biochemical, and genetic investigations have led to substantial recent revisions in conventional thinking about how the products of this pathway are synthesized. The most notable remaining gap in knowledge of the phenylpropanoid pathway is C3H.
Certain intermediates of phenylpropanoid pathway are precursors for lignin. In a parallel manner, in the last decade, our understanding of lignin biosynthesis has rapidly progressed. In many cases, the genetic manipulation of genes encoding enzymes of the conventional lignin pathway has generated unexpected results which have led the scientific community to re-evaluate lignin biosynthesis. The analysis of transgenics and mutants have demonstrated that genetically modified lignins may possess significant advantages over and above traditional raw materials currently used in the pulp and paper industry. In order to further “fine-tune” lignin profiles in economically important plant species in a rational manner, new biotechnological strategies must be employed. Thus, it is also desired to identify novel target genes in the biosynthesis of lignin by molecular and genetic approaches.